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KMID : 1094720200250020293
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Biotechnology and Bioprocess Engineering
2020 Volume.25 No. 2 p.293 ~ p.301
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Promiscuous Trans-splicing Activities Revealed by Next Generation Sequencing-based Analysis of 298 Split Inteins
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Seo Han-Na
Bang Du-Hee
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Abstract
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Protein trans-splicing is a naturally occurring process in which two protein fragments are ligated by a reaction between two intein domains, called split inteins. Despite their usefulness in research, the reactivity and structure of only a few split inteins have been studied. We used cell-based kanamycin selection and next-generation sequencing (NGS) to simultaneously measure the splicing reactivity of 298 N-intein?C-intein combinations derived from the DnaE gene of cyanobacteria. Additionally, we confirmed the splicing activities by measuring the growth of cells individually harboring each split intein under kanamycin selection. Overall, the N-intein?C-intein combinations were promiscuous in their trans-splicing activities, although certain combinations did not splice actively. These results and the NGS-based analysis in this research would be helpful for the development of novel split inteins and further understanding of the trans-splicing mechanism.
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KEYWORD
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high-throughput screening, intein, NGS, protein engineering
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